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Lipid dynamics in boar sperm studied by advanced fluorescence imaging techniques

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Abstract

The (re)organization of membrane components is of special importance to prepare mammalian sperm to fertilization. Establishing suitable methods to examine physico-chemical membrane parameters is of high interest. We characterized the behavior of fluorescent (NBD) analogs of sphingomyelin (SM), phosphatidylserine (PS), and cholesterol (Ch) in the acrosomal and postacrosomal macrodomain of boar sperm. Due to their specific transverse membrane distribution, a leaflet-specific investigation of membrane properties is possible. The behavior of lipid analogs in boar sperm was investigated by fluorescence lifetime imaging microscopy (FLIM), fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS). The results were compared with regard to the different temporal and spatial resolution of the methods. For the first time, fluorescence lifetimes of lipid analogs were determined in sperm cell membrane and found to be in a range characteristic for the liquid-disordered phase in artificial lipid membranes. FLIM analyses further indicate a more fluid microenvironment of NBD-Ch and NBD-PS in the postacrosomal compared to the acrosomal region. The concept of a more fluid cytoplasmic leaflet is supported by lower fluorescence lifetime and higher average D values (FCS) for NBD-PS in both head compartments. Whereas FLIM analyses did not indicate coexisting distinct liquid-ordered and -disordered domains in any of the head regions, comparisons between FRAP and FCS measurements suggest the incorporation of NBD-SM as well as NBD-PS in postacrosomal subpopulations with different diffusion velocity. The analog-specific results indicate that the lipid analogs used are suitable to report on the various physicochemical properties of different microenvironments.

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References

  • Adkins EM, Samuvel DJ, Fog JU, Eriksen J, Jayanthi LD, Vaegter CB, Ramamoorthy S, Gether U (2007) Membrane mobility and microdomain association of the dopamine transporter studied with fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. Biochemistry 46:10484–10497

    Article  CAS  PubMed  Google Scholar 

  • Almeida PF, Pokorny A, Hinderliter A (2005) Thermodynamics of membrane domains. Biochim Biophys Acta 1720:1–13

    Article  CAS  PubMed  Google Scholar 

  • Amaro M, Sachl R, Jurkiewicz P, Coutinho A, Prieto M, Hof M (2014) Time-resolved fluorescence in lipid bilayers: selected applications and advantages over steady state. Biophys J 107:2751–2760

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Axelrod D, Koppel DE, Schlessinger J, Elson E, Webb WW (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16:1055–1069

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Boerke A, Tsai PS, Garcia-Gil N, Brewis IA, Gadella BM (2008) Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane: functional relationship with zona binding and the zona-induced acrosome reaction. Theriogenology 70:1188–1196

    Article  CAS  PubMed  Google Scholar 

  • Bou Khalil M, Chakrabandhu K, Xu H, Weerachatyanukul W, Buhr M, Berger T, Carmona E, Vuong N, Kumarathasan P, Wong PT, Carrier D, Tanphaichitr N (2006) Sperm capacitation induces an increase in lipid rafts having zona pellucida binding ability and containing sulfogalactosylglycerolipid. Dev Biol 290:220–235

    Article  PubMed  Google Scholar 

  • Braga J, Desterro JM, Carmo-Fonseca M (2004) Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes. Mol Biol Cell 15:4749–4760

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chattopadhyay A (1990) Chemistry and biology of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: fluorescent probes of biological and model membranes. Chem Phys Lipid 53:1–15

    Article  CAS  Google Scholar 

  • Christova Y, James P, Mackie A, Cooper TG, Jones R (2004) Molecular diffusion in sperm plasma membranes during epididymal maturation. Mol Cell Endocrinol 216:41–46

    Article  CAS  PubMed  Google Scholar 

  • Daleke DL (2003) Regulation of transbilayer plasma membrane phospholipid asymmetry. J Lipid Res 44:233–242

    Article  CAS  PubMed  Google Scholar 

  • de Almeida RF, Loura LM, Prieto M (2009) Membrane lipid domains and rafts: current applications of fluorescence lifetime spectroscopy and imaging. Chem Phys Lipids 157:61–77

    Article  PubMed  Google Scholar 

  • Eckford PD, Sharom FJ (2005) The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids. Biochem J 389:517–526

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Elson EL (2001) Fluorescence correlation spectroscopy measures molecular transport in cells. Traffic 2:789–796

    Article  CAS  PubMed  Google Scholar 

  • Fery-Forgues S, Fayet JP, Lopez A (2003) Drastic changes in the fluorescence properties of NBD probes with the polarity of the medium: involvement of a TICT state? J Photochem Photobiol A Chem 70:229–243

    Article  Google Scholar 

  • Fuchs B, Jakop U, Goritz F, Hermes R, Hildebrandt T, Schiller J, Müller K (2009) MALDI-TOF “fingerprint” phospholipid mass spectra allow the differentiation between Ruminantia and Feloideae spermatozoa. Theriogenology 71:568–575

    Article  CAS  PubMed  Google Scholar 

  • Gadella BM, Miller NG, Colenbrander B, van Golde LM, Harrison RA (1999) Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: elimination of labeling artifacts. Mol Reprod Dev 53:108–125

    Article  CAS  PubMed  Google Scholar 

  • Greube A, Müller K, Töpfer-Petersen E, Herrmann A, Müller P (2001) Influence of the bovine seminal plasma protein PDC-109 on the physical state of membranes. Biochemistry 40:8326–8334

    Article  CAS  PubMed  Google Scholar 

  • Guo L, Har JY, Sankaran J, Hong Y, Kannan B, Wohland T (2008) Molecular diffusion measurement in lipid bilayers over wide concentration ranges: a comparative study. ChemPhysChem 9:721–728

    Article  CAS  PubMed  Google Scholar 

  • Haldar S, Chattopadhyay A (2013) Application of NBD-labeled lipids in membrane and cell biology. In: Mely Y, Duportail G (eds) Fluorescent methods to study biological membranes. Springer, Berlin, pp 37–50

    Google Scholar 

  • Haupts U, Maiti S, Schwille P, Webb WW (1998) Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. Proc Natl Acad Sci USA 95:13573–13578

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Howes EA, Hurst SM, Jones R (2001) Actin and actin-binding proteins in bovine spermatozoa: potential role in membrane remodeling and intracellular signaling during epididymal maturation and the acrosome reaction. J Androl 22:62–72

    CAS  PubMed  Google Scholar 

  • Ikawa M, Inoue N, Benham AM, Okabe M (2010) Fertilization: a sperm’s journey to and interaction with the oocyte. J Clin Invest 120:984–994

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Im JS, Yu KO, Illarionov PA, LeClair KP, Storey JR, Kennedy MW, Besra GS, Porcelli SA (2004) Direct measurement of antigen binding properties of CD1 proteins using fluorescent lipid probes. J Biol Chem 279:299–310

    Article  CAS  PubMed  Google Scholar 

  • James PS, Wolfe CA, Ladha S, Jones R (1999) Lipid diffusion in the plasma membrane of ram and boar spermatozoa during maturation in the epididymis measured by fluorescence recovery after photobleaching. Mol Reprod Dev 52:207–215

    Article  CAS  PubMed  Google Scholar 

  • James PS, Hennessy C, Berge T, Jones R (2004) Compartmentalisation of the sperm plasma membrane: a FRAP, FLIP and SPFI analysis of putative diffusion barriers on the sperm head. J Cell Sci 117:6485–6495

    Article  CAS  PubMed  Google Scholar 

  • Jamil K (1984) Plasma membrane cytoskeletal complex of the mammalian spermatozoa. Arch Androl 13:177–193

    Article  CAS  PubMed  Google Scholar 

  • Jones R, James PS, Howes L, Bruckbauer A, Klenerman D (2007) Supramolecular organization of the sperm plasma membrane during maturation and capacitation. Asian J Androl 9:438–444

    Article  CAS  PubMed  Google Scholar 

  • Jones R, Howes E, Dunne PD, James P, Bruckbauer A, Klenerman D (2010) Tracking diffusion of GM1 gangliosides and zona pellucida binding molecules in sperm plasma membranes following cholesterol efflux. Dev Biol 339:398–406

    Article  CAS  PubMed  Google Scholar 

  • Julien M, Tournier JF, Tocanne JF (1993) Differences in the transbilayer and lateral motions of fluorescent analogs of phosphatidylcholine and phosphatidylethanolamine in the apical plasma membrane of bovine aortic endothelial cells. Exp Cell Res 208:387–397

    Article  CAS  PubMed  Google Scholar 

  • Kang M, Day CA, Drake K, Kenworthy AK, DiBenedetto E (2009) A generalization of theory for two-dimensional fluorescence recovery after photobleaching applicable to confocal laser scanning microscopes. Biophys J 97:1501–1511

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kang M, Day CA, Kenworthy AK, DiBenedetto E (2012) Simplified equation to extract diffusion coefficients from confocal FRAP data. Traffic 13:1589–1600

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kawano N, Yoshida K, Miyado K, Yoshida M (2011) Lipid rafts: keys to sperm maturation, fertilization, and early embryogenesis. J Lipid 2011:264706

    Article  Google Scholar 

  • Klein AS, Schaefer M, Korte T, Herrmann A, Tannert A (2012) HaCaT keratinocytes exhibit a cholesterol and plasma membrane viscosity gradient during directed migration. Exp Cell Res 318:809–818

    Article  CAS  PubMed  Google Scholar 

  • Kol MA, van Dalen A, de Kroon AI, de Kruijff B (2003) Translocation of phospholipids is facilitated by a subset of membrane-spanning proteins of the bacterial cytoplasmic membrane. J Biol Chem 278:24586–24593

    Article  CAS  PubMed  Google Scholar 

  • Kurz A, Viertel D, Herrmann A, Müller K (2005) Localization of phosphatidylserine in boar sperm cell membranes during capacitation and acrosome reaction. Reproduction 130:615–626

    Article  CAS  PubMed  Google Scholar 

  • Ladha S, James PS, Clark DC, Howes EA, Jones R (1997) Lateral mobility of plasma membrane lipids in bull spermatozoa: heterogeneity between surface domains and rigidification following cell death. J Cell Sci 110(Pt 9):1041–1050

    CAS  PubMed  Google Scholar 

  • Lessig J, Gey C, Suss R, Schiller J, Glander HJ, Arnhold J (2004) Analysis of the lipid composition of human and boar spermatozoa by MALDI-TOF mass spectrometry, thin-layer chromatography and 31P NMR spectroscopy. Comp Biochem Physiol B Biochem Mol Biol 137:265–277

    Article  PubMed  Google Scholar 

  • Lin S, Struve WS (1991) Time-resolved fluorescence of nitrobenzoxadiazole-aminohexanoic acid: effect of intermolecular hydrogen-bonding on non-radiative decay. Photochem Photobiol 54:361–365

    Article  CAS  PubMed  Google Scholar 

  • Luconi M, Barni T, Vannelli GB, Krausz C, Marra F, Benedetti PA, Evangelista V, Francavilla S, Properzi G, Forti G, Baldi E (1998) Extracellular signal-regulated kinases modulate capacitation of human spermatozoa. Biol Reprod 58:1476–1489

    Article  CAS  PubMed  Google Scholar 

  • Mackie AR, James PS, Ladha S, Jones R (2001) Diffusion barriers in ram and boar sperm plasma membranes: directionality of lipid diffusion across the posterior ring. Biol Reprod 64:113–119

    Article  CAS  PubMed  Google Scholar 

  • Mukherjee S, Maxfield FR (2004) Membrane domains. Annu Rev Cell Dev Biol 20:839–866

    Article  CAS  PubMed  Google Scholar 

  • Mukherjee S, Kalipatnapu S, Pucadyil TJ, Chattopadhyay A (2006) Monitoring the organization and dynamics of bovine hippocampal membranes utilizing differentially localized fluorescent membrane probes. Mol Membr Biol 23:430–441

    Article  CAS  PubMed  Google Scholar 

  • Müller K, Pomorski T, Müller P, Zachowski A, Herrmann A (1994) Protein-dependent translocation of aminophospholipids and asymmetric transbilayer distribution of phospholipids in the plasma membrane of ram sperm cells. Biochemistry 33:9968–9974

    Article  PubMed  Google Scholar 

  • Müller P, Plazzo AP, Herrmann A (2011) Transbilayer movement and distribution of cholesterol. In: Devaux PF (ed) Membrane asymmetry and transmembrane motion of lipids. Wiley, Hoboken, pp 75–96

    Google Scholar 

  • Nolan JP, Magargee SF, Posner RG, Hammerstedt RH (1995) Flow cytometric analysis of transmembrane phospholipid movement in bull sperm. Biochemistry 34:3907–3915

    Article  CAS  PubMed  Google Scholar 

  • Ostasov P, Sykora J, Brejchova J, Olzynska A, Hof M, Svoboda P (2013) FLIM studies of 22- and 25-NBD-cholesterol in living HEK293 cells: plasma membrane change induced by cholesterol depletion. Chem Phys Lipid 167–168:62–69

    Article  Google Scholar 

  • Parks JE, Lynch DV (1992) Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes. Cryobiology 29:255–266

    Article  CAS  PubMed  Google Scholar 

  • Peterson RN, Gillott M, Hunt W, Russell LD (1987) Organization of the boar spermatozoan plasma membrane: evidence for separate domains (subdomains) of integral membrane proteins in the plasma membrane overlying the principal segment of the acrosome. J Cell Sci 88(Pt 3):343–349

    PubMed  Google Scholar 

  • Pomorski T, Herrmann A, Zachowski A, Devaux PF, Müller P (1994) Rapid determination of the transbilayer distribution of NBD-phospholipids in erythrocyte membranes with dithionite. Mol Membr Biol 11:39–44

    Article  CAS  PubMed  Google Scholar 

  • Pomorski T, Hrafnsdottir S, Devaux PF, van Meer G (2001) Lipid distribution and transport across cellular membranes. Semin Cell Dev Biol 12:139–148

    Article  CAS  PubMed  Google Scholar 

  • Pucadyil TJ, Mukherjee S, Chattopadhyay A (2007) Organization and dynamics of NBD-labeled lipids in membranes analyzed by fluorescence recovery after photobleaching. J Phys Chem B 111:1975–1983

    Article  CAS  PubMed  Google Scholar 

  • Ramstedt B, Slotte JP (2006) Sphingolipids and the formation of sterol-enriched ordered membrane domains. Biochim Biophys Acta 1758:1945–1956

    Article  CAS  PubMed  Google Scholar 

  • Seigneuret M, Zachowski A, Hermann A, Devaux PF (1984) Asymmetric lipid fluidity in human erythrocyte membrane: new spin-label evidence. Biochemistry 23:4271–4275

    Article  CAS  PubMed  Google Scholar 

  • Sengupta P, Hammond A, Holowka D, Baird B (2008) Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles. Biochim Biophys Acta 1778:20–32

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sezgin E, Levental I, Grzybek M, Schwarzmann G, Mueller V, Honigmann A, Belov VN, Eggeling C, Coskun U, Simons K, Schwille P (2012) Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes. Biochim Biophys Acta 1818:1777–1784

    Article  CAS  PubMed  Google Scholar 

  • Simons K, Gerl MJ (2010) Revitalizing membrane rafts: new tools and insights. Nat Rev Mol Cell Biol 11:688–699

    Article  CAS  PubMed  Google Scholar 

  • Stöckl M, Plazzo AP, Korte T, Herrmann A (2008) Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy of fluorescent lipid analogues. J Biol Chem 283:30828–30837

    Article  PubMed Central  PubMed  Google Scholar 

  • Tannert A, Kurz A, Erlemann KR, Müller K, Herrmann A, Schiller J, Töpfer-Petersen E, Manjunath P, Müller P (2007a) The bovine seminal plasma protein PDC-109 extracts phosphorylcholine-containing lipids from the outer membrane leaflet. Eur Biophysi J EBJ 36:461–475

    Article  CAS  Google Scholar 

  • Tannert A, Töpfer-Petersen E, Herrmann A, Müller K, Müller P (2007b) The lipid composition modulates the influence of the bovine seminal plasma protein PDC-109 on membrane stability. Biochemistry 46:11621–11629

    Article  CAS  PubMed  Google Scholar 

  • Tanphaichitr N, Carmona E, Bou Khalil M, Xu H, Berger T, Gerton GL (2007) New insights into sperm-zona pellucida interaction: involvement of sperm lipid rafts. Front Biosci 12:1748–1766

    Article  CAS  PubMed  Google Scholar 

  • Teichmann A, Gibert A, Lampe A, Grzesik P, Rutz C, Furkert J, Schmoranzer J, Krause G, Wiesner B, Schulein R (2014) The specific monomer/dimer equilibrium of the corticotropin-releasing factor receptor type 1 is established in the endoplasmic reticulum. J Biol Chem 289:24250–24262

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • van Gestel RA, Brewis IA, Ashton PR, Helms JB, Brouwers JF, Gadella BM (2005) Capacitation-dependent concentration of lipid rafts in the apical ridge head area of porcine sperm cells. Mol Hum Reprod 11:583–590

    Article  PubMed  Google Scholar 

  • Wang TY, Silvius JR (2000) Different sphingolipids show differential partitioning into sphingolipid/cholesterol-rich domains in lipid bilayers. Biophys J 79:1478–1489

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wang TY, Silvius JR (2003) Sphingolipid partitioning into ordered domains in cholesterol-free and cholesterol-containing lipid bilayers. Biophys J 84:367–378

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wolfe CA, James PS, Mackie AR, Ladha S, Jones R (1998) Regionalized lipid diffusion in the plasma membrane of mammalian spermatozoa. Biol Reprod 59:1506–1514

    Article  CAS  PubMed  Google Scholar 

  • Wustner D (2007) Fluorescent sterols as tools in membrane biophysics and cell biology. Chem Phys Lipid 146:1–25

    Article  Google Scholar 

  • Wustner D, Pomorski T, Herrmann A, Müller P (1998) Release of phospholipids from erythrocyte membranes by taurocholate is determined by their transbilayer orientation and hydrophobic backbone. Biochemistry 37:17093–17103

    Article  CAS  PubMed  Google Scholar 

  • Wustner D, Mukherjee S, Maxfield FR, Müller P, Herrmann A (2001) Vesicular and nonvesicular transport of phosphatidylcholine in polarized HepG2 cells. Traffic 2:277–296

    Article  CAS  PubMed  Google Scholar 

  • Zachowski A (1993) Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement. Biochem J 294(Pt 1):1–14

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the German Research Council (DFG MU1520/2-1) and by the German Ministry of Education and Research (BMBF Number 033 L046). The authors thank Andreas Herrmann and Thomas Korte (Humboldt-University Berlin) for helpful advice and discussion. The authors also appreciate the excellent technical assistance of Anita Retzlaff (Institute for Reproduction of Farm Animals Schönow e.V.) and Jenny Eichhorst (Leibniz Institute for Molecular Pharmacology).

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Authors and Affiliations

  1. Department of Reproduction Biology, Leibniz Institute for Zoo and Wildlife Research (IZW), Alfred-Kowalke-Straße 17, 10315, Berlin, Germany

    Filip Schröter, Ulrike Jakop, Astrid Tannert & Karin Müller

  2. Leibniz Institute for Molecular Pharmacology, Berlin, Germany

    Anke Teichmann & Burkhard Wiesner

  3. Department of Biology, Humboldt-University Berlin, Berlin, Germany

    Ivan Haralampiev & Peter Müller

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  1. Filip Schröter
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  2. Ulrike Jakop
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  3. Anke Teichmann
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Correspondence to Filip Schröter or Karin Müller.

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Schröter, F., Jakop, U., Teichmann, A. et al. Lipid dynamics in boar sperm studied by advanced fluorescence imaging techniques. Eur Biophys J 45, 149–163 (2016). https://doi.org/10.1007/s00249-015-1084-z

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  • DOI: https://doi.org/10.1007/s00249-015-1084-z

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